1. Field of the Invention
The present invention relates to a method for chemical synthesis of oligonucleotides. In particular, the present invention relates to a novel method capable of chemically synthesizing a long-chain DNA or RNA fragment easily and reliably from a base moiety-unprotected nucleotide phosphoroamidite as a unit, as well as to a novel compound used in said method.
2. Description of the Related Art
The phosphoroamidite method is used most widely at present as a method of chemically synthesizing oligonucleotides such as DNA fragments and RNA fragments (Nucleic Acid Research, 17:7059-7071, 1989). In general, this phosphoroamidite method makes use of a condensation reaction between a nucleoside phosphoroamidite and a nucleoside as a key reaction using tetrazole as an accelerator. Because this reaction usually occurs competitively on both the hydroxyl group in a sugar moiety and the amino group in a nucleoside base moiety, the selective reaction on only the hydroxyl group in a sugar moiety is required to synthesize a desired nucleotide. Accordingly, the side reaction on the amino group was prevented in the prior art by protecting the amino group, as illustrated in the following reaction scheme: ##STR2##
However, the protective group should be removed when synthesis was finished, and operationally complicated organic reactions and a large amount of expensive and harmful reagents are required to introduce and remove said protective group, which in view of practical usability, economical efficiency, environmental protection etc., is a great problem in carrying out this prior method. Accordingly, there is demand for a method of chemically synthesizing an oligonucleotide from an amino group-unprotected nucleoside phosphoroamidite as a unit, and the method of Letsinger et al., as shown in the following reaction scheme, is known as a pioneering method (Nucleic Acids Research, 20:1879-1882, 1992): ##STR3##
However, the method of Letsinger et al. is not practical, not universal and is not used in practice since there are following disadvantages:
(1) condensation yield in each step is low (about 97%: at least 99% yield is required for synthesis of a 50-mer or more long-chain oligonucleotide) and a commercial automatic DNA synthesizer cannot be used for this method, so a long-chain oligonucleotide consisting of 50 to 100 nucleotides generally required in chemical synthesis of DNA etc. cannot be synthesized;
(2) highly reactive, specific nucleoside phosphoroamidites only can be used, and thus this method has a limited scope of application and is not practical; and
(3) pyridine hydrochloride used as an accelerator is an unstable compound with very high moistureproofness, and thus its handling is difficult.